Diabetes affects 25.8 million individuals in the USA and at least 387 million individuals worldwide. Type 2 diabetes (T2D) accounts for more than 90% of diabetes cases. Metformin has been used to treat T2D since the 1950s, and now over 150 million people worldwide take this medication. In 2012, in guidelines for the treatment of T2D, the American Diabetes Association and the European Association for the Study of Diabetes jointly recommended metformin as the initial drug to prescribe to T2D. Yet how metformin acts remains only partially understood and controversial. We find that low metformin concentrations typically found in the portal vein suppress glucose production in primary hepatocytes through the activation of AMPK. However, it remains unclear which isoform of the AMPK? subunits is critical for the suppression of glucose production in hepatocytes by metformin, and metformin binding sites on AMPK subunits have not been characterized yet. Using liver-specific AMPK?1, AMPK?2, and combined AMPK?1/2 knockout mice and mutations of AMPK subunits, we will address these questions in Aim 1. Over one-third of diabetic patients exhibit various degrees of metformin resistance. We find that activation of the cAMP-PKA pathway, a hallmark of patients with diabetes mellitus, directly antagonizes AMPK activation by phosphorylating AMPK? at S485, which in turn reduces AMPK enzymatic activity. This could result in metformin resistance. We propose to study the mechanism leading to metformin resistance in Aim 2, which has important clinical implications for metformin usage in diabetic patients. Salicylate, a commonly used agent, is known to activate AMPK. We will test salicylate's effect on the improvement of metformin efficacy in the suppression of glucose production in Aim 3. We believe this work will provide new evidence for understanding the mechanism of metformin's action and new therapeutic guidelines for the use of metformin to treat T2D.